Project Summary/Abstract Lung vascular network and related pulmonary circulation are essential for both cardiovascular and respiratory functions. Developmental abnormalities of the pulmonary circulation contribute to several neonatal and pediatric cardiopulmonary diseases. However, the exact developmental processes of pulmonary vasculature and circulation remain highly controversial. Earlier study using vascular casting and electron microscopy suggests that simultaneous and independent proximal angiogenesis and peripheral vasculogenesis occur in early embryonic mouse lung, and pulmonary circulation is established later on by connecting these two structures. But other works using serial 3-D reconstruction of human embryonic lung or endothelium-specific reporter mice suggest that the pulmonary circulation arises by either the process of vasculogenesis or angiogenesis exclusively. One major obstacle to limit these studies is lack of genetic tools for respectively tracking the pulmonary vascular cells of different origins. We have generated a unique transgenic mouse line, in which embryonic lung mesenchymal cells can be specifically marked from the beginning of lung organogenesis. Using this system, our preliminary studies suggest that lung mesenchyme-derived vascular endothelial cells (vasculogenesis) and supporting cells can be distinguished from those derived from pre- existing endothelial cells of extrapulmonary origin (angiogenesis), and that lung mesenchyme-derived endothelial cells can coalesce into pre-existing vessels of extrapulmonary origin in early embryonic mouse lung, and then become the major source of the growing vasculature in both proximal and distal parts of fetal lung at mid-gestation. Thus, we hypothesize that extrapulmonary angiogenic vessels initially serve as a stem, and trigger nearby lung mesenchymal progenitor cell differentiation into vascular endothelial and supporting cells, which in turn coalesce into the pre-existing vessels, and become the major source for subsequent pulmonary circulation network development. Two specific aims are proposed to determine (1) the origins of pulmonary vascular endothelial and supporting cells, (2) the dynamic processes of pulmonary circulation establishment. Using the Tbx4 lung enhancer-driven Tet-On inducible Cre/fluorescence protein reporter mice, the origin(s) and the dynamic growth of vascular endothelial cells (angiogenesis vs. vasculogenesis) and vascular supporting cells will be determined in vivo. By intra-cardiac perfusion of fluorescence-lectin to label the connected lung endothelial cells, the initiation of pulmonary circulation will also be defined. Thus, this self- contained small project will reveal a new concept that embryonic lung mesenchymal progenitor cells contribute to pulmonary vascular network formation via angiogenesis and/or vasculogenesis. The obtained data will significantly advance our knowledge in lung organogenesis and related diseases.